Antioxidant and Anti-Inflammatory Effects of Scoparia dulcis L .

Abstract

Different extracts were obtained from Scoparia dulcis L. (Scrophulariaceae) by successive extraction with hexane, chloroform, and methanol. These extracts exhibited significant antioxidant capacity in various antioxidant models mediated (xantine oxidase and lipoxygenase) or not mediated (2,2-diphenyl-picrylhydrazyl, ferric-reducing antioxidant power, β-carotene bleaching, lipid peroxidation) by enzymes. The antioxidant activity of the extracts was related to their phytochemical composition in terms of polyphenol and carotenoid contents. The chloroform extract was richest in phytochemicals and had the highest antioxidant activity in the different antioxidant systems. All the extracts exhibited less than 50% inhibition on xanthine oxidase but more than 50% inhibition on lipid peroxidation and lipoxygenase. The extracts strongly inhibited lipid peroxidation mediated by lipoxygenase.

Introduction

S coparia dulcis L. (Scrophulariaceae), commonly known as sweet broomweed, is a perennial herb harboring white flowers. This plant is common in wastelands and fallow fields of tropical and subtropical regions, where it is well known as a medicinal plant with medical power.¹

Ethnobotanical investigations carried out in these regions found that the plant is used to help with the symptoms of several diseases, such as arterial hypertension² and diabetes mellitus,³ that are related to inflammation and oxidative stress.^4,5

Many medicinal properties of S. dulcis have been previously demonstrated, including its antidiabetic, anti-inflammatory, and antioxidant activities in vivo;^6
–8 its potent inhibition of 2,2-diphenyl-picrylhydrazyl (DPPH) radicals;⁹ and its effect on lipid peroxidation in fowl egg yolk.¹⁰

Furthermore, a few phenolic and terpenic compounds have been isolated from S. dulcis and tested for various biological activities.^11

–14

Reactive oxygen species are responsible for damage to phospholipids in biomembranes by a process known as lipid peroxidation¹⁵ that can be mediated enzymatically or nonenzymatically.¹⁶

Enzymatically mediated lipid peroxidation is related to enzymes such as lipoxygenase and xanthine oxidase, which are linked to inflammatory processes where their activation generates lipid peroxidation products.¹⁷ Lipid peroxidation has been implicated in aging and in various diseases: atherosclerosis, cataract, rheumatoid arthritis, and neurodegenerative disorders such as Alzheimer disease.^16,18

However, it is becoming clear that for in vitro and in vivo characterization of antioxidant propensities, no one method can comprehensively predict antioxidant efficacy. So, the use of more than one method is recommended, and extrapolating the in vitro data should be done more cautiously.^19
–21

This study reports the biological activities of S. dulcis in several antioxidative and anti-inflammatory in vitro models. The composition in polyphenols and carotenoids was also investigated, along with their contribution to the reported antioxidant and anti-inflammatory properties.

Materials and Methods

Plant material

Scoparia dulcis L. (whole plant) was collected at Gampela (25 km east of Ouagadougou, Burkina Faso). Taxonomic identification was verified by the Laboratoire de Biologie et Ecologie Végétales (University of Ouagadougou, Burkina Faso), where a voucher specimen (SD_{-ca 001}) has been deposited and archived.

Extraction

Air-dried grounded Scoparia dulcis (25 g, whole plant) was sequentially extracted with 250 mL of hexane, chloroform, and methanol by using a Soxhlet apparatus. The extracts were then concentrated to dryness in a vacuum evaporator and stored for the different investigations.

Chemicals

All chemicals were of analytical grade. Allopurinol, aluminium trichloride (AlCl₃), bovine serum albumin, β-carotene type I, Folin–Ciocalteu reagent, gallic acid, lipoxygenase type I-B from soybean, quercetin, sodium phosphate dibasic (Na₂HPO₄), sodium phosphate monobasic (NaH₂PO₄), sodium carbonate (Na₂CO₃), thiobarbituric acid, α-tocopherol type V from vegetable oil, xanthine oxidase from bovine milk, and xanthine (2.6-dihydroxypurine) were purchased from Sigma-Aldrich. DPPH, potassium persulfate, and trichloroacetic acid were supplied by Fluka Chemika. Ascorbic acid, potassium hexacyanoferrate K₃Fe(CN)₆, and iron trichloride (FeCl₃) were provided by Labosi.

Quantification of polyphenols

Total polyphenols were determined by following the Folin–Ciocalteu method as described by Singleton et al. ²² Each extract was dissolved in a minimum volume of the solvent used for the extraction and then suspended in distilled water. The diluted aqueous solution of each extract (0.5 mL, 0.1 mg/mL) was mixed with Folin–Ciocalteu reagent (0.2 N, 2.5 mL). This mixture was allowed to stand at room temperature for 5 minutes, and then sodium carbonate solution (2 mL, 75 g/L in water) was added. After 2 hours of incubation in darkness, the absorbance was measured at 760 nm against a water blank. A standard calibration curve was plotted by using gallic acid (0–200 mg/L; R² =0.9996). The results were expressed as mg of gallic acid equivalents per 100 mg of extract (GAE/100 mg).

The total flavonoids were estimated as described by Arvouet-Grant et al. ²³ A diluted methanolic solution of each extract (2 mL, 0.1 mg/mL) was mixed with a solution (2 mL) of aluminium trichloride (2% in methanol). The absorbance was read at 415 nm after 10 minutes against a blank sample consisting of a methanol extract (2 mL) and plant extract (2 mL). Quercetin was used as reference compound to produce the standard curve, and the results are expressed as mg quercetin equivalents per 100 mg of extract (QE/100 mg).

β-carotene and lycopene content

β-carotene and lycopene were determined as described by Barros et al. ²⁴ Dried extract (100 mg) was vigorously shaken with 10 mL of acetone–hexane mixture (4:6) for 1 minute and filtered through Whatman filter paper. The absorbance of the filtrate was then measured at 453, 505, and 663 nm. Contents of β-carotene and lycopene were calculated according to the following equations: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}Lycopene (mg / 100 mL) & = -0.0458 {\rm A}_{663} + 0.372 {\rm A}_{505} & \\ & \quad -0.0806{\rm A}_{453} \\ \beta-{carotene(mg/100 mL) } & = 0.216 {\rm A}_{663}-0.304{\rm A}_{505} \\& \quad + 0.452 {\rm A}_{453} \end{align*}\end{document}

The results were expressed as mg of carotenoid/g of extract.

DPPH radical scavenging

The ability of the extract to scavenge the 2.2-diphenyl-1-picrylhydrazyl radical was evaluated as described by Vélazquez et al. ²⁵

Briefly, a freshly prepared DPPH solution (1.5 mL, 20 mg/mL in methanol) was added to plant extract (0.75 mL, 10 μg/mL in methanol). After shaking, the mixture was incubated for 15 minutes in darkness at room temperature; absorbance was then measured at 517 nm against a blank. The antioxidant content was determined by using a standard curve of ascorbic acid (0–10 μg/mL; R² =0.9999). The results were expressed as mmol ascorbic acid equivalent antioxidant content per g of extract (AAE/g). Ascorbic acid was used as positive control.

Ferric-reducing antioxidant power assay

The Fe (III) to Fe (II) reducing power was evaluated as described by Hinneburg et al. ²⁶ Briefly, each extract solution (1 mL, 0.1 mg/mL in phosphate buffer) was mixed with 2.5 mL of phosphate buffer (0.2 M; pH, 6.6) and 2.5 mL of potassium hexacyanoferrate (1% in water) solution. After 30-minute incubation at 50°C, 2.5 mL of trichloroacetic acid (10% in water) was added and the mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant (2.5 mL) was mixed with 2.5 mL of water and 0.5 mL of aqueous FeCl₃ (0.1%), then absorbance was read at 700 nm. Ascorbic acid was used to generate the calibration curve. The reducing power was expressed as AAE/g. Ascorbic acid was used as positive control.

β-carotene linoleate assay

The antioxidant activity of S. dulcis extracts was evaluated by the β-carotene linoleate model system as described by Barros et al.,²⁴ with some modifications. A solution of β-carotene (0.5 mL, 0.8 mg/mL) was pipetted into a 100-mL round-bottom flask. After the chloroform was removed, 47 μL of linoleic acid, 362 μL of Tween 40 emulsifier, and 100 mL of distilled water were added to the flask and shaken vigorously. Aliquots (4.8 mL) of this emulsion were transferred into different test tubes containing 0.2 mL of extract solution (100 μg/mL). The tubes were shaken and incubated at 50°C in a water bath. As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm by using a spectrophotometer. Absorbances were then recorded at 20-minute intervals until the control sample had changed color. A blank, devoid of β-carotene, was prepared for background subtraction. Antioxidant activity was calculated by using the following equation: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} \hbox{Antioxidant activity} & = (\beta \hbox{\rm - carotene content after 2 hours } \\ & \quad \hbox{of assay/initial} \ \beta-{\rm carotene} \\ & \quad {\rm content}) \times 100. \end{align*}\end{document}

α-Tocopherol was used as standard.

Inhibition of lipid peroxidation in rat liver homogenate

The inhibition effect of S. dulcis extract on lipid peroxidation was determined by measuring malondialdehyde formation according to the thiobarbituric acid method as described by Su et al. ²⁷ FeCl₂-H₂O₂ was used to induce the rat liver (Wistar rats, 155–201 g) homogenate peroxidation. In this method, 0.2 mL of S. dulcis extract (1.5 mg/mL in Tris-HCl buffer 20 mM; pH, 7.4) was mixed with 1 mL of 1% liver homogenate (in Tris-HCl buffer 20 mM; pH, 7.4); 50 μL of FeCl₂ (0.5 mM) and H₂O₂ (0.5 mM) was then added. The mixture was incubated at 37°C for 60 minutes, and then 1 mL of trichloroacetic acid (15%) and thiobarbituric acid (0.67%) was added. The mixture was incubated in boiled water for 15 minutes. After centrifugation (2000 rpm for 5 minutes using the ALC Centrifugette 4206 [Milan, Italy]), the absorbance of the supernatant was recorded at 532 nm. Ascorbic acid was used as the positive control. The percentage of inhibition effect was calculated according to the following equation: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{\rm Inhibition} (\%) = [1 - (A_1 - A_2) / A_0] \times 100\end{align*}\end{document}

A ₀ is the absorbance of the control (without extract), A ₁ is the absorbance of the extract addition, and A ₂ is the absorbance without liver homogenate.

Inhibition of xanthine oxidase

The xanthine oxidase inhibitory activity was assayed on a CECIL spectrophotometer as described by Owen and Timothy,²⁸ with some modifications. The assay mixture consisted of 150 μL of phosphate buffer (0.066 M; pH, 7.5), 50 μL of extract solution (1 mg/mL, in phosphate buffer), and 50 μL of enzyme solution (0.28 U/mL). After preincubation at room temperature (25°C) for 3 minutes, the reaction was initiated by the addition of 250 μL of substrate solution (xanthine, 0.15 M in the same buffer). A blank without enzyme solution was also prepared. The reaction was monitored for 3 minutes at 295 nm and velocity (V₀) was recorded. Phosphate buffer was used as negative control (activity of the enzyme without extract solution). Allopurinol was used as positive control.

The percentage of xanthine oxidase inhibition was calculated with the following equation: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{\rm Inhibition} \ (\%) = (V_{0 \ control} - V_{0 \ sample}) \times 100 / V_{0 \ control} \end{align*}\end{document}

V _{0 control} is the activity of the enzyme in absence of the extract solution, and V _{0 sample} is the activity of the enzyme in presence of the extract solution or allopurinol.

Inhibition of lipoxygenase

The inhibition of soybean lipoxygenase type 1-B was assayed spectrophotometrically as described by Maiga et al.,²⁹ with some modifications. Briefly, 200 μL of the enzyme solution (200 U/mL) was prepared in boric acid buffer (0.2 M; pH, 9.0) and mixed with 50 μL of extract solution (1 mg/mL in boric acid buffer) and then incubated at room temperature for 3 minutes. The reaction was then initiated by the addition of 250 μL of substrate solution (linoleic acid, 250 μM), and the velocity was recorded for 90 seconds at 234 nm. Boric acid buffer was used as negative control (activity of the enzyme without extract solution). Quercetin was used as positive control. The percentage of lipoxygenase inhibition was calculated according to the previous equation used for xanthine oxidase.

Statistical analysis

All the reactions were performed in triplicate, and data are presented as mean±standard deviation. Data were analyzed by 1-way analysis of variance followed by the Tukey multiple-comparison test. Analyses were done by using XLSTAT7.1 software. A P value less than .05 was used as the criterion for statistical significance.

Results

Yield of extraction

The yield of each extract was obtained by calculating the ratio of the dried extract by the weight of the initial powder of the plant material. Methanol extract showed the highest yield (15.12%), followed by hexane extract (4.74%) and chloroform extract (2.98%).

Polyphenol and carotenoid composition

Total polyphenols and flavonoids (polyphenols), as well as β-carotene and lycopene (carotenoids), were quantified (Table 1) from sequential extracts (hexane, chloroform, methanol) of S. dulcis. As shown, total polyphenol contents varied according to the polarity of the extraction solvent. This was not seen for total flavonoids, lycopene, and β-carotene. Hence, the highest amount of total polyphenols (22.20±1.27 mg GAE/100 mg) was recovered in methanol, whereas flavonoids (12.70±0.94 mg QE/100 mg), β-carotene (5.35±0.08 mg/g), and lycopene (0.63±0.01 mg/g) were mostly extracted in chloroform. Content of β-carotene was approximately 10 times higher than that of lycopene in all the extracts except methanol extract.

Table 1.

Phytochemical Composition of Scoparia dulcis extracts

	Extracts
Phytochemical	Hexane	Chloroform	Methanol
Total polyphenols (mg GAE/100 mg)	6.02±1.41^a	13.56±1.58^b	22.20±1.27^c
Flavonoids (mg QE/100 mg)	5.12±1.28^a	12.70±0.94^a	8.44±0.31^b
β-carotene (mg/g)	1.44±0.05^b	5.35±0.08^c	0.32±0.08^a
Lycopene (mg/g)	0.10±0.01^a	0.63±0.01^b	0.28±0.02^b

Values are expressed as means±standard deviation (n=3 experiments).

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Values within each line with different superscripted letters differ significantly (P>.05) as determined by using analysis of variance.

GAE, gallic acid equivalents; QE, quercetin equivalents.

Antioxidant capacity

The antioxidant capacity of S. dulcis extracts was evaluated through DPPH, FRAP, β-carotene, and lipid peroxidation models (Table 2), each assay involving a different antioxidant mechanism.

Table 2.

Antioxidant Capacity of Scoparia dulcis Extracts

	Antioxidant assays
Extract	DPPH (mmol AAE/g)	FRAP (mmol AAE/g)	β-carotene (%)	Liver lipid peroxidation (%)
Hexane	0.03±0.006^a	0.29±0.01^b	41.10±1.66^a	68.03±3.44^ab
Chloroform	0.71±0.03^c	1.57±0.14^c	48.29±1.40^c	62.42±1.04^a
Methanol	0.75±0.01^c	0.96±0.01^a	40.23±2.22^a	73.33±1.04^c
Ascorbic acid	9.65±0.74	5.70±0.15	ND	48.75±1.28
α-Tocopherol	ND	ND	45.30±2.39^c	ND

Values are expressed as mean values±standard deviation (n=3 experiments). DPPH and FRAP activities are given in mmol AAE/g (10⁻³ mol ascorbic acid per g) of extract. β-carotene bleaching and lipid peroxidation activities are expressed as inhibitory percentage at, respectively, 100 μg/mL and 1.5 mg/mL, as initial concentrations of the extracts. Ascorbic acid (1.5 mg/mL) and α-tocopherol (50 μg/mL) were used as standards.

abc

Values within each column with different superscripted letters differ significantly (P>.05) as determined by using analysis of variance.

AAE, ascorbic acid; ND, not determined.

FRAP ranged from 0.29±0.01 mmol AAE/g for hexane extract to 1.57±0.14 mmol AAE/g for chloroform extract. DPPH radical–scavenging activity was 0.75±0.019 mmol AAE/g for methanol extract, 0.71±0.03 mmol AAE/g for chloroform extract, and 0.03±0.006 mmol AAE/g for hexane extract.

According to the β-carotene model, none of the extracts (100 μg/mL) reached 50% inhibition. The chloroform extract had the best inhibitory activity (48.29%±1.40%) of β-carotene bleaching, followed by methanol extract and hexane extract (40.23%±2.22% and 41.10%±1.66%, respectively). At this dose (100 μg/mL), the chloroform activity was similar to 50 μg/mL of the standard α-tocopherol (45.30%±2.39%).

The chloroform extract, which is the richest in flavonoids and carotenoids, exhibited the best antioxidant activity in DPPH, FRAP, and β-carotene assays. Ascorbic acid used as positive control had a 4-fold reducing power (5.7±0.15 mmol AAE/g) and a 13-fold scavenging activity compared with chloroform extract.

The inhibition percentages of liver lipid peroxidation ranged from 62.42%±1.04% for chloroform extract to 73.33%±1.42% for methanol extract at a concentration of 1.5 mg/mL (corresponding to a final concentration of 90.91 μg/mL). Methanol extract, which showed the highest content of total polyphenols, also exhibited the highest inhibition of liver lipid peroxidation. At this concentration, the inhibition percentages of all extracts were greater than 50% and were higher than those for ascorbic acid (48.75%±1.28%) used as positive control. Methanol extract was further tested at different concentrations on lipid peroxidation (Table 3) alongside the measurement of ascorbic acid. As shown, methanol extract and ascorbic acid inhibited lipid peroxidation in a dose-dependent manner. Concentration that inhibits 50% of lipid peroxidation (IC₅₀) was 0.46±0.02 mg/mL for methanol extract and 1.56±0.15 mg/mL for ascorbic acid. These results show the significant activity of S. dulcis extracts against lipid peroxidation on rat liver homogenate.

Table 3.

Dose-Dependent Inhibition of Lipid Peroxidation

	Inhibition of lipid peroxidation (%)
Concentration (mg/mL)	Methanol extract	Ascorbic acid
0.3125	44.10±1.60	30.51±3.54
0.625	52.56±1.17	36.30±0.71
1.25	59.48±1.77	41.47±2.21
1.5	73.33±1.42	48.75±1.28
2.5	95.12±1.93	68.54±4.69
5	94.45±2.30	75.89±0.97
IC₅₀ (mg/mL)	0.46±0.02	1.56±0.15

Values are expressed as mean values±standard deviation (n=3 experiments). Inhibitory activities of lipid peroxidation by a methanol extract of Scoparia dulcis in varying concentrations alongside ascorbic acid as positive control.

IC₅₀, initial concentrations that inhibit 50% of lipid peroxidation.

In vitro lipoxygenase and xanthine oxidase inhibition

Anti-inflammatory activities were evaluated through xanthine oxidase and lipoxygenase inhibitions. All the extracts of S. dulcis (100 μg/mL) exhibited a weak to moderate inhibitory activity (7.96%±1.76% to 31.56%±2.22%) on xanthine oxidase and showed a strong effect (70.57%±0.78% to 94.5%8±0.90%) on lipoxygenase (Table 4). Hence, all the extracts inhibited lipoxygenase more than 50% and displayed levels higher than quercetin (52.74%±1.72%) used as positive control. Chloroform extract, which contained the highest amount of flavonoids and carotenoids (Table 1), was the most active in both enzymatic models.

Table 4.

Inhibitory Activities of Scoparia dulcis on Xanthine Oxidase and Lipoxygenase

	Inhibitory activities (%)
Extract	Xanthine oxidase	Lipoxygenase
Hexane	7.96±1.76^a	91.66±2.06^b
Chloroform	31.56±2.22^b	94.58±0.90^b
Methanol	28.31±4.05^b	70.57±0.78^a
Allopurinol	96.42±0.60	ND
Quercetin	75.11±4.53	52.74±1.72

Values are expressed as mean values±standard deviation (n=3 experiments). Allopurinol (100 μg/mL) and quercetin (50 μg/mL) were used as standards.

For each column, values followed by the same superscripted letter are not statistically different (P>0.05) as determined by using analysis of variance.

ND, not determined.

Discussion

This paper describes the characterization of differently prepared S. dulcis extracts in several antioxidative models.

Lipoxygenases are enzymes involved in inflammatory processes and oxidative stress because their activation generates lipid peroxidation products and the biosynthesis of inflammatory lipid mediators.¹⁷ Lipoxygenase inhibition and β-carotene bleaching assay are methods based on the same substrate that is linoleate, considered the model substrate because it is the most abundant polyunsaturated fatty acids in vivo.¹⁶ In the β-carotene bleaching assay, the antioxidants in the different extracts of S. dulcis act by neutralizing free radicals from linoleate; in contrast, they prevent free radical generation by inhibiting lipoxygenase action on linoleate or by often scavenging similar radicals generated within the active site of the enzyme.^30,31 Accordingly, at the same concentration (100 μg/mL), all the extracts were more efficient in lipoxygenase inhibition (they all reached 50% inhibition) than in β-carotene bleaching assay (none of them reached 50% inhibition).

This finding may indicate that S. dulcis extracts more efficiently prevent the generation of free radicals from linoleate than they neutralize free radicals once they are produced. In addition, the more an extract is active in lipoxygenase inhibition, the more it is also in β-carotene bleaching assay; however, the levels of inhibition of the different extracts were not significantly different between the 2 activities. The extracts from nonpolar solvents that are hexane extract and chloroform extract (with highest content in β-carotene) showed the best inhibitory activity on both lipoxygenase and β-carotene bleaching as previously reported.^29,32 That is in accordance with previous studies reporting that the lipid peroxidation in the β-carotene emulsion system was inhibited by apolar antioxidants because their hydrophobicity enhanced their activity, shielding the emulsion by concentrating at the lipid air surface.³² Thus, the antioxidant activity in β-carotene bleaching assay of S. dulcis extracts may be attributed to lipophilic carotenoids and mainly β-carotene, which is known to be a potent antioxidant that quenches the singlet oxygen and other free radicals.¹⁵ This antioxidant activity of S. dulcis extracts in β-carotene system is similar to that of Melissa officinalis and Laurus nobilis essential oils and decoctions at the same concentration³³ and to that of a methanol extract of Agaricus arvensis at 5 mg/mL.²⁴

The liver plays a vital role in regulating various physiologic processes. Therefore, the liver is a target for numerous hepatotoxic agents that cause oxidative damages and alter its principal functions.³⁴ Much of the liver damage is induced by lipid peroxidation.³⁵ Although lipid peroxidation is particularly active in biomembranes as liver tissue rich in various polyunsaturated fatty acids, all the extracts of S. dulcis inhibited it (all of them reached 50% inhibition) to a greater extent than did β-carotene bleaching with only linoleate as substrate. This finding suggests the diversity of antioxidant compounds in S. dulcis extracts, which are able to neutralize various thiobarbituric acid reactive substances produce by biomembrane oxidation. Iron ion (Fe²⁺), a known potent pro-oxidant agent in biological systems, was used in this assay to initiate the liver lipid peroxidation, which is further propagated by peroxyl and alkoxyl radicals.³⁶ The extracts could prevent the initiation of peroxidation process by reducing iron ion or by neutralizing the free radicals produced within the propagation phase. Polyphenols inhibit the oxidation by direct scavenging of lipid alkoxyl and peroxyl radicals involved in the propagation phase.³⁷ Polyphenols in S. dulcis extracts may have a strong contribution to its anti-lipoperoxidation effect. This activity of a water extract of S. dulcis was previously reported in fowl egg yolk;¹⁰ it is noteworthy that methanol extract of S. dulcis efficiently inhibited Fe²⁺-induced lipid peroxidation in liver (IC₅₀, 0.46±0.02 mg/mL) more so than did the water extract in fowl egg yolk (IC₅₀, 1.08 mg/mL)¹⁰ but less than its inhibition of 2,2-azobis(2-amidinopropane) dihydrochloride–induced lipid peroxidation by other methanolic extracts of Nepalese medicinal plants.³⁰ Other previous in vivo studies confirmed the capacity of S. dulcis extract to significantly decrease thiobarbituric acid reactive substances produced within liver and kidney lipid oxidation in streptozotocin diabetic rats.⁸ This hepatoprotective activity against biomembrane oxidation in liver demonstrated by S. dulcis extracts can be helpful for the management of liver disorders.

Therefore, the liver antilipid peroxidation of S. dulcis extracts did not correlate with lipoxygenase inhibition as suggested in previous studies³⁰ because each extract of S. dulcis was more effective in lipoxygenase inhibition despite lesser activity in liver lipid peroxidation assay. Thus, S. dulcis extracts are more effective inhibitors of lipid peroxidation mediated by lipoxygenase, and this pathway could explain the markedly in vivo anti-inflammatory effect of ethanol extract of S. dulcis in rats as previously studied.⁷

No direct relationship was found between lipoxygenase inhibition of S. dulcis extracts and their antioxidant activity by using FRAP and DPPH methods, in accordance with prior studies.^38,39 Indeed, lipoxygenase inhibition is an enzymatic method that differs from the nonenzymatic antioxidant methods (FRAP, DPPH) with other mechanisms including scavenging, quenching, and removal of active oxidants.¹⁶ The scavenging activity of DPPH radicals by S. dulcis extract was previously reported as an inhibitory percentage of DPPH of about 27.5% at 10 mg/mL,⁹ that differs from the DPPH-scavenging activity observed in the present study. Therefore, the ability to scavenge the DPPH radical was strongly related to lipid peroxidation⁴⁰ and polyphenol content,⁴¹ and the extracts of S. dulcis are in this scheme. In addition, the extract obtained from methanol, a well-known polyphenol extraction solvent,⁴² was more efficient on both DPPH radical (0.75±0.01 mmol AAE/g) and lipid peroxidation (73.33%±1.04%) than the other extracts. Then, the inhibitor compounds of lipid peroxidation and DPPH radicals might mostly be polyphenols, which are known to often act in synergy with other antioxidants as β-carotene.⁴³

Polyphenols and carotenoids might contribute to inhibitory activities of xanthine oxidase and lipoxygenase. Indeed, flavonoids are well-known xanthine oxidase and lipoxygenase inhibitors.^31,44 Phenolic structures in general have the potential to strongly interact with proteins and act as antioxidants by inhibiting some enzymatic proteins involved in radical generation, such as lipoxygenases and xanthine oxidase.⁴⁵

Conclusion

The 3 extracts of S. dulcis obtained from increasing-polarity solvents exhibited significant levels of various antioxidant and anti-inflammatory capacity, which may be due to its composition of polyphenols and carotenoids.

The extracts exerted their antioxidant properties through different mechanisms, including via the enzymatic pathway by inhibition of lipoxygenase and xanthine oxidase, 2 enzymes involved in inflammatory processes and lipid peroxidation. In these in vitro antioxidant systems, the different extracts of S. dulcis prevented lipid peroxidation (mediated by lipoxygenase) better than it was able to repair the damage from lipid peroxidation (in the β-carotene system). Thus, S. dulcis may have a hepatoprotective role and a beneficial effect on diseases characterized by inflammation and lipid peroxidation.

Footnotes

Acknowledgment

We are grateful to the International Atomic Energy Agency (Vienna, Austria) for providing the basic equipments of the laboratory through the Technical Cooperation Project BKF 5002.

Author Disclosure Statement

No competing financial interests exist.

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